Methods and compositions for inducing immune responses against clostridium difficile

ABSTRACT

Disclosed herein are methods and compositions for treating or preventing bacterial infection. In particular, the methods and compositions are directed towards C. difficile infection. In particular aspects, the compositions are vaccines containing multimeric polypeptides containing portions of multiple toxins from bacteria. The polypeptides induce effective immune responses thus treating or preventing infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.62/471,636, filed Mar. 15, 2017 and 62/474,434, filed Mar. 21, 2017, thedisclosures of which are each incorporated herein for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:NOVV_058_02WO_SeqList_ST25, date recorded: Mar. 13, 2018, file size 187kilobytes).

BACKGROUND

Vaccination against disease using a subunit-based vaccine is dependenton producing sufficient amounts of the protein antigen and maintainingstability of the antigen such that the protein remains effective whenadministered to a target population.

Complications in producing subunit vaccines arise at multiple stepsduring production. The target protein can be produced at low levels, orcan be insoluble, resulting in economically-unfavorable production, evenwhen the protein had particularly favorable immunogenicity profile.

Bacterial infections remain a health concern. Indeed, bacterial vaccinesare increasingly sought after as bacteria evolve resistance tofront-line antibiotics. Bacterial subunit vaccines rely on recombinantprotein production. However, bacterial proteins can often be difficultto produce at high level due to low expression, and insolubility, andthey can also suffer from reduced stability. Better approaches toproducing vaccines, particularly for difficult antigen targets, wouldthus provide global health benefits. In particular, infection byclostridial bacteria, notably C. difficile remains a particular problem.Clostridium difficile infection (CDI) is the leading cause of nosocomialantibiotic-associated diarrhea in developed countries. Hypervirulentstrains have evolved causing severe disease with increased mortality.Homologous glucosylating toxins, TcdA and TcdB, and binaryADP-ribosylating toxin (CDT) are major virulence factors causingpathogenesis. There is an unmet need for vaccines targeting thesetoxins.

SUMMARY OF THE INVENTION

Disclosed herein are methods and compositions of inducing immuneresponses against C. difficile. The compositions contain polypeptidescontaining multiple C. difficile toxins, which, when administered to asubject, induce advantageous immune responses. Methods for producing themulti-toxin polypeptides are also disclosed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, C. difficile triple toxin vaccine constructs. Figure showsillustration of C. diff triple toxin vaccine containing the bindingdomains of CDTb, Tcd B, and Tcd A with (construct 1420) and without(construct 1470) a furin cleavage site after the activation domain ofCDTb.

FIG. 2. Expression and solubility of triple toxin vaccine BV1470 andBV1420. Spodoptera frugiperda Sf9 insect cells were infected at a MOI of0.1 with recombinant baculovirus BV1420 and BV1470, harvested at 48 and72 hours postinfection, and analyzed for protein expression by SDS-PAGEand coomassie staining. An equal volume of total protein (T, cells andmedium) and clarified medium (M) were mixed with 2×SDS-PAGE samplebuffer and run on a 4%-12% polyacrilamide NuPage gel. Pelleted, infectedcells were solubilized in 1% NP9, 25 mM Tris, 50 mM NaCL, pH 8.0 buffer.Lysed cells were centrifuged at 9000×g for 40 min. Supernatant (5,soluble) was removed, pellet (I, insoluble) was suspended in buffer tooriginal volume, and analyzed by SDS-PAGE as described above. Locationof triple toxin protein marked with an arrow.

FIG. 3. Time course expression of triple toxin vaccine BV1470 andBV1420. Spodoptera frugiperda Sf9 insect cells were infected withrecombinant baculovirus BV1420 and BV1470, as described in FIG. 6. Totalprotein, medium, soluble, and insoluble protein was analyzed by SDS-PAGEand coomassie staining at various timepoints postinfection. Location oftriple toxin protein is marked with an arrow.

FIG. 4. Purification of triple toxin vaccine. The triple toxin vaccinewas purified from total cell culture of infected Sf9 cells following theaddition of NP9 to a final concentration of 0.2%. NP9 extract wasclarified twice and purified on consecutive Fractogel EMD TMAE, PhenylHP, and Source 30Q columns. The triple toxin was eluted from each columnand loaded onto the next column as shown. Eluted triple toxin positivefractions from the Source 30Q column were pooled and filter sterilizedthrough a 0.2 μM filter.

FIG. 5. Purification of triple toxin vaccine BV1470 from Sf9 cells.Triple toxin vaccine BV1470 was purified from infected cells asdescribed in FIG. 8. Final filtered product from the Source 30Q columnwas analyzed for purity by SDS-PAGE and coomassie staining. Triple toxinprotein was identified by western blot using anti-CDTb, anti-TcdB, andantiTcdA antibodies.

FIG. 6. Purification of triple toxin vaccine BV1420 from Sf9 cells.Triple toxin vaccine BV1420 was purified from infected cells asdescribed in FIG. 8. Final filtered product from the Source 30Q columnwas analyzed for purity by SDS-PAGE and coomassie staining. Triple toxinprotein was identified by western blot using anti-TcdB antibodies.

FIG. 7. Particle size distribution by volume graph for triple toxinBV1420. Particle size of triple toxin BV1420 was determined by dynamiclight scattering using a Zeta Sizer Nano. Graph of size distribution byvolume is shown.

FIG. 8. Particle size distribution by intensity graph for triple toxinBV1470. Particle size of triple toxin BV1420 was determined by dynamiclight scattering using a Zeta Sizer Nano. Graph of size distribution byintensity is shown.

FIGS. 9A-9D. Electronmicrographs of negative stained triple toxinBV1420. Electron-micrograph of purified triple toxin BV1420 was dilutedto approximately 10 ug/ml and negatively stained with uranyl acetate.

FIG. 10. BV1420 triple toxin vaccine mouse lethal toxin challengestudy 1. Mice were immunized on day zero and day 14 with triple toxinvaccine BV1420 and challenged on day 35 with a lethal dose of Tcd A orCDT and monitored for 10 days post challenge. Mice were bleed as shownand serum analyzed for anti-toxin IgG and for toxin neutralizingantibodies. Animals were monitored for mortality and morbidity for 10days after toxin challenge.

FIG. 11. BV1420 triple toxin vaccine mouse lethal toxin challenge study1—serum anti-toxin IgG responses. Day 42 serum samples were assayed forAnti-Tcd A, anti-Tcd B, and anti-CDT IgG titers by ELISA using nativetoxins bound to plates.

FIG. 12. BV1420 triple toxin vaccine mouse lethal toxin challenge study1—toxin neutralizing antibody (TNA) titers. Toxin neutralization titerswere determined using a colorimetric Vero cell based assay. Titerindicated are the reciprocal of the highest dilution of serum that didnot kill cells.

FIG. 13. BV1420 triple toxin vaccine mouse lethal toxin challenge study1—animal survival. Animal survival was determined 10 days postchallenge. Animals showing greater than 20% weight loss were sacrificedand recorded as dead.

FIG. 14. BV1420 triple toxin vaccine mouse lethal toxin challenge study2—toxin B survival. Mice were immunized on day zero and day 14 withtriple toxin vaccine BV1420 and challenged on day 35 with a lethal doseof Tcd B and monitored for 10 days post challenge. Mice were bled asshown and serum analyzed for anti-toxin NG and for toxin neutralizingantibodies (TNA). Animals were monitored for mortality and morbidity for10 days after toxin challenge.

FIG. 15. BV1420 triple toxin vaccine mouse lethal toxin challenge study2—anti-toxin IgG levels. Day 42 serum samples were assayed for Anti-TcdA, anti-Tcd B, and anti-CDT IgG titers by ELISA using native toxinsbound to plates.

FIG. 16. BV1420 triple toxin vaccine mouse lethal toxin challenge study2—toxin B TNA titers. Toxin neutralization titers were determined usinga colorimetric Vero cell based assay. Titer indicated are the reciprocalof the highest dilution of serum that did not kill cells.

FIG. 17. BV1420 triple toxin vaccine mouse lethal toxin challenge study2—toxin B survival. Animal survival was determined 10 days postchallenge. Animals showing greater than 20% weight loss were sacrificedand recorded as dead.

FIG. 18. Additional vaccine proteins with the TcdB gene translocationdomain are shown. BV1512 is shown in the bottom diagram.

FIG. 19. Mulitimer Protein Expression: Expression and western blotanalysis of multimer protein BV1512.

FIG. 20. Quadrivalent Multimer Protein Expression: FIG. 25 illustratestwo quadrivalent multimer proteins. In both cases, a peptide from asecond TcdB strain is introduced to broaden immunity against multiplestrains. In the upper diagram, the TcdB peptide from Strain 027 is addedat the C-terminus. In the lower diagram, the peptide is introducedbetween the TcdB protein and the TcdA(R19) protein from the firststrain, strain 630.

FIG. 21. Quadrivalent Multimer Protein Expression: Expression andwestern blot analysis of the quadrivalent protein shown in the upperdiagram of FIG. 20.

FIG. 22. Quadrivalent Multimer Protein Expression: Expression andwestern blot analysis of the quadrivalent protein shown in the lowerdiagram of FIG. 20.

FIG. 23. C. difficile Toxins and Design of Chimeric Trivalent (T) andQuadravalent (Q) Toxin Fusion Proteins. FIG. 23A shows the illustrationof the functional domains of C. difficile toxin A (TcdA), toxin B(TcdB), and binary toxin (CDT) used to construct the chimeric trivalentand quadravalent toxin fusion proteins. TcdA and TcdB share commonfunctional domains including the enzymatic glucosyltransferase (GT)domain, autocatalytic cysteine protease (CP) domain, pore-formingtranslocation (PT) domain (orange), and receptor binding domain (RBD).The binary toxin (CDT) consists of the enzymatic ADP-ribosyltransferasecomponent (CDTa) and receptor binding component (CDTb). CDTb contains a42 amino acid (aa) signal sequence with two serine-type proteolyticcleavage sites (arrow) which, when cleaved, generates a 20 kDa and 75kDa fragment. FIG. 24B shows the illustration of the chimeric trivalenttoxin fusion protein (T-toxin) and a chimeric quadravalent toxin fusionprotein (Q-toxin). The T-toxin fusion protein consists of thefull-length coding sequence for CDTb with the RBD of TcdB₍₀₀₃₎containing 24 repeats and the truncated RBD of TcdA with 19 repeats. Theexpressed T-toxin fusion protein consists of 1813 aa with a molecularweight (MW) of 205 kDa. The Q toxin fusion protein consists of thefull-length coding sequence for CDTb to the RBD of TcdB₍₀₀₃₎ containing24 repeats, the RBD of TcdA truncated at 19 repeats, and the RBD ofTddB₍₀₂₇₎ containing 24 repeats. The expressed Q-toxin fusion proteinconsists of 2359 aa with a molecular weight of 268 kDa.

FIGS. 24A-24C. Expression and Purification of T-Toxin and Q-Toxin FusionProteins. SDS-PAGE of purified T-toxin (lanes 2 and 3) migrates with amolecular weight of 205 kDa and Q-toxin (lanes 4 and 5) migrates with amolecular weight of 268 kDa. Molecular weight marker (lane 1). FIG. 24Ashows T-toxin and Q-toxin purity was >90% as determined by SDS-PAGEscanning densitometry. FIG. 24B shows western blot analysis as probedwith rabbit anti-CDTb specific antibodies. FIG. 24C shows western blotanalysis as probed with chicken anti-TcdB specific antibodies. FIG. 24Dshows western blot analysis as probed with chicken anti-TcdA specificantibodies.

FIGS. 25A-25C. Immunogenicity of T-Toxin and Q-Toxin Fusion Proteins inMice. Groups of female C57BL/6 mice (N=10/group) were immunized 1M onDays 0 and 14 with toxin (100 ug) or Q-toxin (100 μg) adjuvanted withalum (50 μg), or PBS (control group). Serum was collected 18 days afterthe second vaccination. FIG. 25A shows serum IgG titers to TcdA,Tcd₍₀₀₃₎, and CDTb determined by ELISA. FIG. 25B showstoxin-neutralizing antibody titers for each toxin determined in the Verocell assay. In FIG. 25C, Mice received a lethal dose (MLD_(100%)=2.0 μg)of TcdB₍₀₀₃₎ administered IP 21 days after the second immunization.*Significance was determined by Mantel-Cox log-rank test comparing theI-toxin or Q-toxin groups to the PBS control group.

FIGS. 26A-26D. Immunogenicity of T-Toxin and Q-Toxin Fusion Proteins inHamsters. Male hamsters (N=8/group) were immunized IM 3 times at 21-dayintervals with 30 μg Q-toxin adjuvanted with 120 μg alum, or PBS(control group). Two weeks after the third dose, samples were collectedand analyzed. FIG. 26A shows serum IgG titers to TcdA, TcdB₍₀₀₃₎, andCDTb determined by ELISA. FIG. 26B shows toxin-neutralizing antibodytiters for each toxin determined in the Vero cell assay. In FIGS. 26Cand 26D, two weeks after the third immunization, all animals weretreated with clindamycin (10 mg/kg) IP one day prior to spore challengeand were challenged by gavage with 200 cfu C. difficile strain 630 (C)or with 500 cfu C. difficile strain B/NAP1/027 (D). Animals wereobserved for 8 days post challenge.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “adjuvant” refers to a compound that, when usedin combination with an immunogen, augments or otherwise alters ormodifies the immune response induced against the immunogen. Modificationof the immune response may include intensification or broadening thespecificity of either or both antibody and cellular immune responses.

As used herein, the terms “immunogen,” “antigen,” and “epitope” are usedinterchangeably and refer to substances such as proteins, and peptidesthat are capable of eliciting an immune response.

As used herein, the term “fusion protein” means a protein comprised oftwo or more proteins or protein fragments that are joined or fused,directly or indirectly via a linking peptide, at the amino terminus ofone protein and the carboxy terminus of another protein, to form asingle continuous polypeptide. In some aspects, a fusion protein may bereferred to as a “multivalent protein.” A multivalent protein containsproteins or protein fragments from two or more three discrete proteinantigens that are fused together.

The terms “treat,” “treatment,” and “treating,” as used herein, refer toan approach for obtaining beneficial or desired results, for example,clinical results. For the purposes of this invention, beneficial ordesired results may include inhibiting or suppressing the initiation orprogression of an infection or a disease; ameliorating, or reducing thedevelopment of, symptoms of an infection or disease; or a combinationthereof.

“Prevention,” as used herein, is used interchangeably with “prophylaxis”and can mean complete prevention of an infection or disease, orprevention of the development of symptoms of that infection or disease;a delay in the onset of an infection or disease or its symptoms; or adecrease in the severity of a subsequently developed infection ordisease or its symptoms.

As used herein an “effective dose” or “effective amount” refers to anamount of an immunogen sufficient to induce an immune response thatreduces at least one symptom of malaria. An effective dose or effectiveamount may be determined e.g., by measuring amounts of neutralizingsecretory and/or serum antibodies, e.g., by plaque neutralization,complement fixation, enzyme-linked immunosorbent (ELISA), ormicroneutralization assay.

As used herein, the term “vaccine” refers to a preparation including animmunogen (e.g. a fusion protein described herein) derived from apathogen, which is used to induce an immune response against thepathogen that provides protective immunity (e.g., immunity that protectsa subject against infection with the pathogen and/or reduces theseverity of the disease or condition caused by infection with thepathogen). The protective immune response may include formation ofantibodies and/or a cell-mediated response. Depending on context, theterm “vaccine” may also refer to a suspension or solution of animmunogen that is administered to a vertebrate to produce protectiveimmunity.

As used herein, the term “subject” includes humans and other animals.The subject, in one embodiment, is a human.

As used herein, the term “pharmaceutically acceptable” means beingapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia, European Pharmacopeia or othergenerally recognized pharmacopeia for use in mammals, and moreparticularly in humans. These compositions can be useful as a vaccineand/or antigenic compositions for inducing a protective immune responsein a vertebrate.

As used herein, the term “about” means plus or minus 10% of theindicated numerical value.

Overview

The present disclosure provides methods and compositions for achievinghigh expression of large proteins, particularly multivalent proteinscontaining multiple antigens, from insect cells. The production of highlevels of proteins as disclosed herein is particularly unexpected inview of prior experiences in the field.

Multivalent Proteins

The multivalent (the multivalent protein may also be referred to hereinas a multimer) proteins disclosed herein can protect against multiplepathogens and/or the effects from multiple pathogenic proteins from thesame organism. For example, certain pathogens may produce multiplemolecules that each negatively affects a subject. A more effectiveresponse is produced by inducing responses against multiple separateantigens.

The proteins multivalent protein contains protein portions from multiplebacterial toxins the In some aspects, the multivalent protein comprises,or consists of, portions of proteins from the same organism, such astoxins for example. In other aspects, the multivalent protein comprises,or consists of, proteins from more than one organism. In particularaspects, no two proteins of a multivalent protein are from the sameorganism. In some aspects, the same proteins from different strains(i.e., isologs) may be used to produce the portion. Using the sameprotein from a different strain allows protection against multiplestrains and is particularly useful in situations where virulent strainsnewly arise. Other examples include C. botulinum, which has 8serological types, A through H. The methods and compositions disclosedherein can be used to provide a single vaccine against all 8 serotypes.Other particular examples include combination toxin vaccines to protectagainst cholera, diptheria and shigella, or tetanus, purtussis anddiptheria. Thus, in some aspects, a multimeric protein may containportions from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different proteins. Theportions may be used as components to produce the multimeric immunogenicpolypeptides.

Exemplary multimers and components used to produce vaccines aredescribed in the table below. Nucleic acid sequences encoding Q-toxinand BV1512, as well as alternative nucleic acid sequences for BV1420 andBV1470, are those using standard codon conversion appropriate degeneratecodons that encode the indicated amino acid.

Protein Nucleic Acid Vaccine Construct Components Sequence SequenceBV1420 CDTb SEQ ID NO: 10 SEQ ID NO: 14 (SEQ ID NO: 9; TcdB SEQ ID NO:11 SEQ ID NO: 15 SEQ ID NO: 13) TcdA SEQ ID NO: 12 SEQ ID NO: 16 BV1470CDTb SEQ ID NO: 2 SEQ ID NO: 6 (SEQ ID NO: 1; TcdB SEQ ID NO: 3 SEQ IDNO: 7 SEQ ID NO: 5) TcdA SEQ ID NO: 4 SEQ ID NO: 8 BV1512 CDTb SEQ IDNO: 18 (SEQ ID NO: 17) TD SEQ ID NO: 19 TcdAR19 SEQ ID NO: 20 Q-toxinCDTb SEQ ID NO: 22 (SEQ ID NO: 21) TcdB003 SEQ ID NO: 23 TcdA SEQ ID NO:24 TcdB027 SEQ ID NO: 25

Additional vaccine constructs may use the various components above indifferent orientations. In addition, proteins having at least 90%identity to each of these disclosed sequences may be used as componentsto produce a multimer protein.

Linkers

In some aspects, linkers may be used between one or more proteins in themultivalent proteins. In some aspects, the linker is a poly-(Gly)nlinker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19,or 20. In other aspects, the linker is GG, GGG, or GGGG (SEQ ID NO: 26).In yet other aspects, the linker is selected from the group consistingof: dipeptides, tripeptides, and quadripeptides. Preferred dipeptidesare Alanine-Serine (AS), Leucine-Glutamic acid (LE), Serine-Arginine(SR).

Multivalent antigens are particularly suited for protection againstorganisms that release multiple toxins into a subject. For example,bacteria are known to produce toxins that cause disease in humans. Thus,while the primary focus of the disclosure is C. difficile; themultimeric polypeptides of the disclosure may be prepared using portionsof protein toxins from other species.

Toxin-producing species include C. perfringes, C. botulinum, C.difficile, and C. tetani), Bacillus (e.g., B. anthracis), Vibrio (e.g.,Vibrio cholerae), Shigella, and Corynebacterium. C. difficile releasestwo enteric toxins, A and B, which are produced by toxigenic strains.Toxin A is an enterotoxin with minimal cytotoxic activity, whereas toxinB is a potent cytotoxin but has limited enterotoxic activity. A thirdtoxin, Binary Toxin, also known as CDT, is also produced by thebacteria. Sequences encoding toxin A and B are known (Moncrief et al.,Infect. Immun. 65:1105-1108 (1997); Barroso et al., Nucl. Acids Res.18:4004 (1990); Dove et al. Infect. Immun. 58:480-488 (1990)). Sequencesencoding Binary Toxin are also known (Accession Nos. ABS57477, AAB67305,AAF81761).

The usefulness of the present disclosure for protection against pathogeninfection is illustrated by a trivalent protein vaccine against C.difficile. FIG. 1 shows the structure of two exemplary multimer proteins(BV1420 and BV1470). Each multimer contains portions of three toxinproteins, Toxin A (TcdA), Toxin B (TcdB), and binary toxin (CDTb), fromC. difficile. Triple toxin 1420 also contains a furin cleavage site.These proteins are large—over 1800 amino acids—and would not bepreviously have been expected to yield usable amounts of protein whenexpressed in insect cells. Surprisingly, however, both proteins areexpressed at high levels. See FIG. 3. Indeed, as FIG. 5 demonstrates,the yield for BV1470 was 269 mg/L. Similarly, the yield for BV1420 was166 mg/L.

Analysis of the purified multimer proteins confirmed they were innanoparticle structures with peak diameters around about 16 nm forBV1420 and about 18 nm for BV1470. Notably, the distribution ofdiameters shown in FIGS. 7 and 8 illustrates that a high percentage ofthe multimer proteins retained nanoparticle structure afterpurification.

Administering the BV1420 trivalent nanoparticles to mice demonstratesthat immune responses to all three proteins were obtained. Moreover, asFIG. 13 illustrates the immune response obtained protected 100% of micefrom lethal challenge with Toxin A and Binary toxin, as well as 67% to83% of mice in response to lethal challenge with Toxin B. In contrast,mice in the PBS control group all died, with the exception of two micein the binary toxin control group.

Quadrivalent toxins are also a preferred type of multimer immunogenicpeptide. FIG. 20 shows two illustrative examples with four portions orcomponents arranged in sequence. Despite the substantial length of themultimer, good protein production was obtained. FIG. 22.

FIG. 23 illustrates the conversion of a ti-toxin fusion protein to aquadrivalent toxin by addition of portion of a toxin from a second TcdBtype. Comparing these two proteins shows that insect cell expression isable to give high level production. See FIG. 24A-D.

Thus, exemplary multimers include portions organized in variousorientation. For example, starting from the N-terminus the first portionmay be a TcdA portion, a TcdB portion or a CDTb portion. The secondportion may be a TcdA portion, a TcdB portion or a CDTb portion. Thethird portion may be a TcdA portion, a TcdB portion or a CDTb portion.The fourth portion, if present, may be a TcdA portion, a TcdB portion ora CDTb portion. Thus, each portion may occupy each position. Typically,though not always, two adjacent portions are not portions from the sametype of toxin. In preferred embodiments, the N-terminal portion is a aCDTb portion.

Molecular Biology Techniques

The multivalent proteins disclosed herein are prepared through molecularbiology approaches. General texts which describe molecular biologicaltechniques, which are applicable to the present invention, such ascloning, mutation, cell culture and the like, include Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook el al.,Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”) andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (“Ausubel”). These texts describemutagenesis, the use of vectors, promoters and many other relevanttopics related to, e.g., the cloning and mutating PfCSP, etc. Thus, theinvention also encompasses using known methods of protein engineeringand recombinant DNA technology to improve or alter the characteristicsof the proteins expressed on or in the fusion proteins of the invention.Various types of mutagenesis can be used to produce and/or isolatevariant nucleic acids that encode for protein molecules and/or tofurther modify/mutate the proteins in or on the fusion proteins of theinvention. They include but are not limited to site-directed, randompoint mutagenesis, homologous recombination (DNA shuffling), mutagenesisusing uracil containing templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

Methods of cloning the proteins are known in the art. A gene can becloned as a DNA insert into a vector. The term “vector” refers to themeans by which a nucleic acid can be propagated and/or transferredbetween organisms, cells, or cellular components. Vectors includeplasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons,artificial chromosomes, and the like, that replicate autonomously or canintegrate into a chromosome of a host cell. A vector can also be a nakedRNA polynucleotide, a naked DNA polynucleotide, a polynucleotidecomposed of both DNA and RNA within the same strand, apoly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, aliposome-conjugated DNA, or the like, that is not autonomouslyreplicating. In many, but not all, common embodiments, the vectors ofthe present invention are plasmids or bacmids.

Thus, the invention comprises nucleotides that encode proteins,including chimeric molecules, cloned into an expression vector that canbe expressed in a cell that induces the formation of fusion proteins ofthe invention. An “expression vector” is a vector, such as a plasmid,that is capable of promoting expression, as well as replication of anucleic acid incorporated therein. Typically, the nucleic acid to beexpressed is “operably linked” to a promoter and/or enhancer, and issubject to transcription regulatory control by the promoter and/orenhancer. In one embodiment, the nucleotides encode for a Plasmodiumprotein (as discussed above). In another embodiment, the expressionvector is a baculovirus vector.

In some embodiments of the invention, proteins may comprise mutationscontaining alterations which produce silent substitutions, additions, ordeletions, e.g., to optimize codon expression for a particular host(change codons in the human mRNA to those preferred by insect cells suchas Sf9 cells). See, for example, U.S. Patent Publication 2005/0118191,herein incorporated by reference in its entirety for all purposes.

In addition, the nucleotides can be sequenced to ensure that the correctcoding regions were cloned and do not contain any unwanted mutations.The nucleotides can be subcloned into an expression vector (e.g.baculovirus) for expression in any cell. The above is only one exampleof how the proteins can be cloned. A person with skill in the artunderstands that additional methods may be used.

Host Cells

The high level expression was obtained in insect cell expressionsystems. Non limiting examples of insect cells are, Spodopterafrugiperda (Sf) cells, e.g. Sf9, Sf21, Trichoplusia ni cells, e.g. HighFive cells, and Drosophila S2 cells.

Vectors, e.g., vectors comprising polynucleotides that encode fusionproteins, can be transfected into host cells according to methods wellknown in the art. For example, introducing nucleic acids into eukaryoticcells can be achieved by calcium phosphate co-precipitation,electroporation, microinjection, lipofection, and transfection employingpolyamine transfection reagents. In one embodiment, the vector is arecombinant baculovirus.

Nanoparticle Production

The nanoparticles may be produced by growing host cells transformed byan expression vector under conditions whereby the recombinant proteinsare expressed. In one aspect, a method of producing a multivalentprotein comprises transfecting vectors encoding the protein into asuitable host cell and expressing the protein under conditions thatallow nanoparticle formation. In another embodiment, the eukaryotic cellis selected from the group consisting of yeast, insect, amphibian, avianor mammalian cells. The selection of the appropriate growth conditionsis within the skill or a person with skill of one of ordinary skill inthe art.

Methods to grow host cells include, but are not limited to, batch,batch-fed, continuous and perfusion cell culture techniques. Cellculture means the growth and propagation of cells in a bioreactor (afermentation chamber) where cells propagate and express protein (e.g.recombinant proteins) for purification and isolation. Typically, cellculture is performed under sterile, controlled temperature andatmospheric conditions in a bioreactor. A bioreactor is a chamber usedto culture cells in which environmental conditions such as temperature,atmosphere, agitation and/or pH can be monitored. In one embodiment, thebioreactor is a stainless steel chamber. In another embodiment, thebioreactor is a pre-sterilized plastic bag (e.g. Cellbag®, Wave Biotech,Bridgewater, N.J.). In other embodiment, the pre-sterilized plastic bagsare about 50 L to 1000 L bags.

Detergent Extraction and Purification of Nanoparticles

The nanoparticles may be harvested from the host cells using detergents.Suitable detergents include non-ionic surfactants. For example, thenon-ionic surfactant may be Bis(polyethylene glycolbis[imidazoylcarbonyl]), nonoxynol-9, Bis(polyethylene glycolbis[imidazoyl carbonyl]), Brij® 35, Brij®56, Brij® 72, Brij® 76, Brij®92V, Brij® 97, Brij® 58P, Cremophor® EL, Decaethyleneglycol monododecylether, N-Decanoyl-N-methylglucamine, n-Decylalpha-Dglucopyranoside,Decyl beta-D-maltopyranoside,n-Dodecanoyl-N-methylglucamide, nDodecyl alpha-D-maltoside, n-Dodecylbeta-D-maltoside, n-Dodecyl beta-D-maltoside,Heptaethylene glycolmonodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethyleneglycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethyleneglycol monododecyl ether, Hexaethylene glycol monohexadecyl ether,Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether, Igepal CA-630,Igepal CA-630,Methyl-6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside,Nonaethyleneglycol monododecyl ether, N-Nonanoyl-N-methylglucamine,N-NonanoylN-methylglucamine, Octaethylene glycol monodecyl ether,Octaethylene glycolmonododecyl ether, Octaethylene glycol monohexadecylether, Octaethylene glycol monooctadecyl ether, Octaethylene glycolmonotetradecyl ether, Octyl-beta-D glucopyranoside, Pentaethylene glycolmonodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethyleneglycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctylether, Polyethylene glycol diglycidyl ether, Polyethylene glycol etherW-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether,Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate,Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl),Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillajabark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85,Tergitol Type 15-S-12, Tergitol Type 15-S-30, Tergitol Type 15-S-5,Tergitol Type 15-S-7, Tergitol Type 15-S-9, Tergitol Type NP-10,Tergitol Type NP-4, Tergitol Type NP-40, Tergitol, Type NP-7 TergitolType NP-9, Tergitol Type TMN-10, Tergitol Type TMN-6,Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecyl ether,Tetraethylene glycol monododecyl ether, Tetraethylene glycolmonotetradecyl ether, Triethylene glycol monodecyl ether, Triethyleneglycol monododecyl ether, Triethylene glycol monohexadecyl ether,Triethylene glycol monooctyl ether, Triethylene glycol monotetradecylether, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, TritonGR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, TritonX-15, Triton X-151, Triton X-200, Triton X-207, Triton® X-100, Triton®X-114, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45,Triton® X-705-70, TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61,TWEEN® 65, TWEEN® 80, TWEEN® 81, TWEEN® 85, Tyloxapol, n-Undecylbeta-D-glucopyranoside, semi-synthetic derivatives thereof, orcombinations thereof. Tergitol NP-9 is a preferred detergent.

Once the host cells have grown for 48 to 72 hours, the cells areisolated from the media and a detergent-containing solution is added tosolubilize the cell membrane, releasing the nanoparticles in a detergentextract. The detergent may be added to a final concentration of about0.1% to about 1.0%. For example, the concentration may be about 0.1%,about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8%, or about1.0%. In certain aspects, the range may be about 0.1% to about 0.3%.Preferably, the concentration is about 0.2%.

The nanoparticles may then be isolated using methods that preserve theintegrity thereof, such as centrifugation. In some aspects, gradientcentrifugation, such as using cesium chloride, sucrose and iodixanol,may be used. Other techniques may be used as alternatives or inaddition, such as standard purification techniques including, e.g., ionexchange and gel filtration chromatography.

In one aspect, the detergent extract is added to multiple columnssequentially. For example, the first column may be an ion chromatographycolumn, such as TMAE, the second column may be a hydrophobic interactioncolumn, such as Phenyl HP, and the third column may be a strong anionexchange column such as a Source 30Q column. Increased purity may beobtained by repeating the three-step procedure.

The following provides a general procedure for making isolating andpurifying proteins. A person of skill in the art would understand thatthere variations that can be utilized

Production is initiated by seeding Sf9 cells (non-infected) into shakerflasks, allowing the cells to expand and scaling up as the cells growand multiply (for example from a 125-ml flask to a 50 L Wave bag). Themedium used to grow the cell is formulated for the appropriate cell line(preferably serum free media, e.g. insect medium ExCell-420, JRH). Next,the cells are infected with recombinant baculovirus at the mostefficient multiplicity of infection (e.g. from about 1 to about 3 plaqueforming units per cell). Once infection has occurred, the fusionproteins (and, optionally, other immunogens) are expressed from thevirus genome. Usually, infection is most efficient when the cells are inmid-log phase of growth (4-8×10⁶ cells/ml) and are at least about 90%viable.

Proteins of the disclosure can be harvested approximately 48 to 96 hourspost infection. In some aspects, harvesting takes place at about 48hours, about 72 hours, or between about 48 and about 72 hours.Typically, harvesting takes place when the levels of VLPs in the cellculture medium are near the maximum but before extensive cell lysis. TheSf9 cell density and viability at the time of harvest can be about0.5×10⁶ cells/ml to about 1.5×10⁶ cells/ml with at least 20% viability,as shown by dye exclusion assay.

To solubilize the particles, directly add Tergitol NP9 to cell cultureto final concentration of 0.2% NP9/25 mM Tris/50 mM NaCl/pH8.0. Incubateat room temperature for 1 hour then centrifuge the lysate at 9000 g for30 min twice. Collected the supernatant containing the nanoparticles.The supernatant is then added to in Buffer A and eluted in Buffer B(Buffer A: 25 mM Tris pH 8.0/50 mM NaCl Buffer B: 25 mM Tris pH 8.0/1MNaCl). The eluate is appled to Phenyl HP columns (Buffer A: 350 mMNa-Citrate/25 mM Tris pH7.5 and Buffer B: 5 mM Tris pH8.0) and then to aSource 30Q column (Buffer A: 25 mM Tris pH8.0/250 mM NaCl Buffer B: 25mM Tris pH8.01M NaCl). The pooled fractions containing the product arepassed through a 2 micron filter. See FIGS. 8-10.

The procedures described above enable a purity of at least about 90%, atleast about 95% or about 98% at a yield of 150 mg/L to about 300 mg/L.Purity may be measured by gel-based approaches that indicate totalprotein.

The intact baculovirus can be inactivated, if desired. Inactivation canbe accomplished by chemical methods, for example, formalin orβ-propiolactone (BPL). Removal and/or inactivation of intact baculoviruscan also be largely accomplished by using selective precipitation andchromatographic methods known in the art, as exemplified above. Methodsof inactivation comprise incubating the sample containing the VLPs in0.2% of BPL for 3 hours at about 25° C. to about 27° C. The baculoviruscan also be inactivated by incubating the sample containing the VLPs at0.05% BPL at 4° C. for 3 days, then at 37° C. for one hour.

The above techniques can be practiced across a variety of scales. Forexample, T-flasks, shake-flasks, spinner bottles, up to industrial sizedbioreactors. The bioreactors can comprise either a stainless steel tankor a pre-sterilized plastic bag (for example, the system sold by WaveBiotech, Bridgewater, N.J.). A person with skill in the art will knowwhat is most desirable for the particular circumstance.

Protein Size and Yield

The yield for the multimer proteins using the methods disclosed hereinis remarkable. In some cases, the yield is about 150 mg/L to about 300mg/L. In some embodiments, the yield is about 40 mg/L, about 60 mg/L,about 80 mg/L, about 100 mg/L, about 150 mg/L, about 200 mg/L, about 250mg/L, or about 300 mg/L. In particular aspects, the yield ranges fromabout 40 mg/L to about 300 mg/L, from about 80 mg/L to about 250 mg/L,or about 100 mg/mL to about 300 mg/L.

Large multimer proteins disclosed herein typically range from about1500-2500 amino acids. In some aspects, they range from about 1500 toabout 2000 amino acids. In other aspects, they range from about 1800 toabout 2000 amino acids.

The multimer proteins form nanoparticles having a typical diameter ofabout 11 nm to about 35 nm. The diameter range may be about 15 nm toabout 25 nm. Illustrative examples of multimer protein nanoparticles inthese ranges are shown in FIG. 9.

Importantly, even though the proteins are large, they remain soluble.For example, the purified multimer protein may be about 80% soluble,about 85% soluble, about 90% soluble, about 95% soluble, about 97%soluble, or about 99° % soluble. In some aspects, solubility is about90% to about 99% or about 90% to about 95%.

Modified Antigens and Polypeptides

The antigens disclosed herein encompass variations and mutants of thoseantigens. In certain aspects, the antigen may share identity to adisclosed antigen. Generally, and unless specifically defined in contextof a specifically identified antigens, the percentage identity may be atleast 80%, at least 90%, at least 95%, at least 97%, or at least 98%.Percentage identity can be calculated using the alignment programClustal Omega, available at www.ebi.ac.uk/Tools/msa/clustalo usingdefault parameters.

In particular aspects, the protein contained in the nanoparticlesconsists of that protein. In other aspects, the protein contained in thenanoparticles comprise that protein. Extensions to the protein itselfmay be for various purposes.

In some aspects, the antigen may be extended at the N-terminus, theC-terminus, or both. In some aspects, the extension is a tag useful fora function, such as purification or detection. In some aspects the tagcontains an epitope. For example, the tag may be a polyglutamate tag, aFLAG-tag, a HA-tag, a polyHis-tag (having about 5-10 histidines), aMyc-tag, a Glutathione-S-transferase-tag, a Green fluorescentprotein-tag, Maltose binding protein-tag, a Thioredoxin-tag, or anFc-tag. In other aspects, the extension may be an N-terminal signalpeptide fused to the protein to enhance expression. While such signalpeptides are often cleaved during expression in the cell, somenanoparticles may contain the antigen with an intact signal peptide.Thus, when a nanoparticle comprises an antigen, the antigen may containan extension and thus may be a fusion protein when incorporated intonanoparticles. For the purposes of calculating identity to the sequence,extensions are not included. In some aspects, the antigen may betruncated. For example, the N-terminus may be truncated by about 10amino acids, about 30 amino acids, about 50 amino acids, about 75 aminoacids, about 100 amino acids, or about 200 amino acids. For example, theC-terminus may be truncated by about 10 amino acids, about 30 aminoacids, about 50 amino acids, about 75 amino acids, about 100 aminoacids, or about 200 amino acids.

Pharmaceutical Compositions

The pharmaceutical compositions disclosed herein comprise a multimerprotein and a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers include any pharmaceutical agent that can beadministered to a subject without undue toxicity, irritation, orallergic reaction. Pharmaceutically acceptable carriers may also includeone or more pharmaceutically acceptable excipient. A pharmaceuticallyacceptable excipient is any excipient that is useful in preparing apharmaceutical composition that is generally safe and non-toxic, and isacceptable for veterinary as well as human pharmaceutical use.

The pharmaceutical compositions useful herein contain a pharmaceuticallyacceptable carrier, including any suitable diluent or excipient, whichincludes any pharmaceutical agent that does not itself induce theproduction of an immune response harmful to the vertebrate receiving thecomposition, and which may be administered without undue toxicity and animmunogen; for example a multimer fusion protein.

In some aspects, formulations may include a pharmaceutically acceptablecarrier or excipient. Pharmaceutically acceptable carriers include butare not limited to saline, buffered saline, dextrose, water, glycerol,sterile isotonic aqueous buffer, and combinations thereof. A thoroughdiscussion of pharmaceutically acceptable carriers, diluents, and otherexcipients is presented in Remington's Pharmaceutical Sciences (MackPub. Co. N.J. current edition). The formulation may be adapted to suitthe mode of administration. In an exemplary embodiment, the formulationis suitable for administration to humans, is sterile, non-particulateand/or non-pyrogenic.

The composition may also contain wetting agents, or emulsifying agents,or pH buffering agents, or mixtures thereof. The composition can be asolid form, such as a lyophilized powder suitable for reconstitution(e.g., with water or saline), a liquid solution, suspension, emulsion,tablet, pill, capsule, sustained release formulation, or powder. Oralformulations may include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, etc.

Adjuvants

The immunogenicity of a particular composition may be enhanced by theuse of non-specific stimulators of the immune response, known asadjuvants. Adjuvants have been used experimentally to promote ageneralized increase in immunity against antigens (e.g., U.S. Pat. No.4,877,611). Immunization protocols have used adjuvants to stimulateresponses for many years, and as such, adjuvants are well known to oneof ordinary skill in the art. Some adjuvants affect the way in whichantigens are presented. For example, the immune response is increasedwhen protein antigens are precipitated by alum. Emulsification ofantigens also prolongs the duration of antigen presentation. Theinclusion of any adjuvant described in Vogel et al., “A Compendium ofVaccine Adjuvants and Excipients (2nd Edition),” herein incorporated byreference in its entirety for all purposes, is envisioned within thescope of this disclosure.

Exemplary adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant. Other adjuvants comprise GMCSP, BCG, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL), MF-59, RIBI, which contains three components extracted frombacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS)in a 2% squalene/Tween® 80 emulsion. In other preferred aspects, Alumsuch as 2% Alhydrogel (Al(OH)₃) is used. In some aspects, the adjuvantmay be a paucilamellar lipid vesicle; for example, Novasomes®.Novasomes® are paucilamellar nonphospholipid vesicles ranging from about100 nm to about 500 nm. They comprise Brij 72, cholesterol, oleic acidand squalene. Novasomes have been shown to be an effective adjuvant(see, U.S. Pat. Nos. 5,629,021, 6,387,373, and 4,911,928.

Saponin Adjuvants

Adjuvants containing saponin may also be combined with the immunogensdisclosed herein. Saponins are glycosides derived from the bark of theQuillaja saponaria Molina tree. Typically, saponin is prepared using amulti-step purification process resulting in multiple fractions. Asused, herein, the term “a saponin fraction from Quillaja saponariaMolina” is used generically to describe a semi-purified or definedsaponin fraction of Quillaja saponaria or a substantially pure fractionthereof.

Saponin Fractions

Several approaches for producing saponin fractions are suitable.Fractions A, B, and C are described in U.S. Pat. No. 6,352,697 and maybe prepared as follows. A lipophilic fraction from Quil A, a crudeaqueous Quillaja saponaria Molina extract, is separated bychromatography and eluted with 70% acetonitrile in water to recover thelipophilic fraction. This lipophilic fraction is then separated bysemi-preparative HPLC with elution using a gradient of from 25% to 60%acetonitrile in acidic water. The fraction referred to herein as“Fraction A” or “QH-A” is, or corresponds to, the fraction, which iseluted at approximately 39% acetonitrile. The fraction referred toherein as “Fraction B” or “QH-B” is, or corresponds to, the fraction,which is eluted at approximately 47% acetonitrile. The fraction referredto herein as “Fraction C” or “QH-C” is, or corresponds to, the fraction,which is eluted at approximately 49% acetonitrile. Additionalinformation regarding purification of Fractions is found in U.S. Pat.No. 5,057,540. When prepared as described herein, Fractions A, B and Cof Quillaja saponaria Molina each represent groups or families ofchemically closely related molecules with definable properties. Thechromatographic conditions under which they are obtained are such thatthe batch-to-batch reproducibility in terms of elution profile andbiological activity is highly consistent.

Other saponin fractions have been described. Fractions B3, B4 and B4bare described in EP 0436620. Fractions QA1-QA22 are described EP03632279B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside(Isconova AB, Ultunaallén 2B, 756 51 Uppsala, Sweden). Fractions QA-1,QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12,QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21, and QA-22of EP 0 3632 279 B2, especially QA-7, QA-17, QA-18, and QA-21 may beused. They are obtained as described in EP 0 3632 279 B2, especially atpage 6 and in Example 1 on page 8 and 9.

The saponin fractions described herein and used for forming adjuvantsare often substantially pure fractions; that is, the fractions aresubstantially free of the presence of contamination from othermaterials. In particular aspects, a substantially pure saponin fractionmay contain up to 40% by weight, up to 30% by weight, up to 25% byweight, up to 20% by weight, up to 15% by weight, up to 10% by weight,up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% byweight, up to 0.5% by weight, or up to 0.1% by weight of other compoundssuch as other saponins or other adjuvant materials.

ISCOM Structures

Saponin fractions may be administered in the form of a cage-likeparticle referred to as an ISCOM (Immune Stimulating COMplex). ISCOMsmay be prepared as described in EP0109942B1, EP0242380B1 and EP0180546B1. In particular embodiments a transport and/or a passenger antigen maybe used, as described in EP 9600647-3 (PCT/SE97/00289).

Matrix Adjuvants

In some aspects, the ISCOM is an ISCOM matrix complex. An ISCOM matrixcomplex comprises at least one saponin fraction and a lipid. The lipidis at least a sterol, such as cholesterol. In particular aspects, theISCOM matrix complex also contains a phospholipid. The ISCOM matrixcomplexes may also contain one or more other immunomodulatory(adjuvant-active) substances, not necessarily a glycoside, and may beproduced as described in EP0436620B1.

In other aspects, the ISCOM is an ISCOM complex. An ISCOM complexcontains at least one saponin, at least one lipid, and at least one kindof antigen or epitope. The ISCOM complex contains antigen associated bydetergent treatment such that that a portion of the antigen integratesinto the particle. In contrast, ISCOM matrix is formulated as anadmixture with antigen and the association between ISCOM matrixparticles and antigen is mediated by electrostatic and/or hydrophobicinteractions.

According to one embodiment, the saponin fraction integrated into anISCOM matrix complex or an ISCOM complex, or at least one additionaladjuvant, which also is integrated into the ISCOM or ISCOM matrixcomplex or mixed therewith, is selected from fraction A, fraction B, orfraction C of Quillaja saponaria, a semipurified preparation of Quillajasaponaria, a purified preparation of Quillaja saponaria, or any purifiedsub-fraction e.g., QA 1-21.

In particular aspects, each ISCOM particle may contain at least twosaponin fractions. Any combinations of weight % of different saponinfractions may be used. Any combination of weight % of any two fractionsmay be used. For example, the particle may contain any weight % offraction A and any weight % of another saponin fraction, such as a crudesaponin fraction or fraction C, respectively. Accordingly, in particularaspects, each ISCOM matrix particle or each ISCOM complex particle maycontain from 0.1 to 99.9 by weight, 5 to 95% by weight, 10 to 90% byweight 15 to 85% by weight, 20 to 80% by weight, 25 to 75% by weight, 30to 70% by weight, 35 to 65% by weight, 40 to 60% by weight, 45 to 55% byweight, 40 to 60% by weight, or 50% by weight of one saponin fraction,e.g. fraction A and the rest up to 100% in each case of another saponine.g. any crude fraction or any other faction e.g. fraction C. The weightis calculated as the total weight of the saponin fractions. Examples ofISCOM matrix complex and ISCOM complex adjuvants are disclosed in U.SPublished Application No. 2013/0129770.

In particular embodiments, the ISCOM matrix or ISCOM complex comprisesfrom 5-99% by weight of one fraction, e.g. fraction A and the rest up to100% of weight of another fraction e.g. a crude saponin fraction orfraction C. The weight is calculated as the total weight of the saponinfractions.

In another embodiment, the ISCOM matrix or ISCOM complex comprises from40% to 99% by weight of one fraction, e.g. fraction A and from 1% to 60%by weight of another fraction, e.g. a crude saponin fraction or fractionC. The weight is calculated as the total weight of the saponinfractions.

In yet another embodiment, the ISCOM matrix or ISCOM complex comprisesfrom 70% to 95% by weight of one fraction e.g., fraction A, and from 30%to 5% by weight of another fraction, e.g., a crude saponin fraction, orfraction C. The weight is calculated as the total weight of the saponinfractions. In other embodiments, the saponin fraction from Quillajasaponaria Molina is selected from any one of QA 1-21.

In addition to particles containing mixtures of saponin fractions, ISCOMmatrix particles and ISCOM complex particles may each be formed usingonly one saponin fraction. Compositions disclosed herein may containmultiple particles wherein each particle contains only one saponinfraction. That is, certain compositions may contain one or moredifferent types of ISCOM-matrix complexes particles and/or one or moredifferent types of ISCOM complexes particles, where each individualparticle contains one saponin fraction from Quillaja saponaria Molina,wherein the saponin fraction in one complex is different from thesaponin fraction in the other complex particles.

In particular aspects, one type of saponin fraction or a crude saponinfraction may be integrated into one ISCOM matrix complex or particle andanother type of substantially pure saponin fraction, or a crude saponinfraction, may be integrated into another ISCOM matrix complex orparticle. A composition or vaccine may comprise at least two types ofcomplexes or particles each type having one type of saponins integratedinto physically different particles.

In the compositions, mixtures of ISCOM matrix complex particles and/orISCOM complex particles may be used in which one saponin fractionQuillaja saponaria Molina and another saponin fraction Quillajasaponaria Molina are separately incorporated into different ISCOM matrixcomplex particles and/or ISCOM complex particles.

The ISCOM matrix or ISCOM complex particles, which each have one saponinfraction, may be present in composition at any combination of weight %.In particular aspects, a composition may contain 0.1% to 99.9% byweight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight,20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40 to 60% byweight, or 50% by weight, of an ISCOM matrix or complex containing afirst saponin fraction with the remaining portion made up by an ISCOMmatrix or complex containing a different saponin fraction. In someaspects, the remaining portion is one or more ISCOM matrix or complexeswhere each matrix or complex particle contains only one saponinfraction. In other aspects, the ISCOM matrix or complex particles maycontain more than one saponin fraction.

In preferred compositions, the saponin fraction in a first ISCOM matrixis Fraction A (a “Fraction A Matrix”) and the saponin fraction in asecond ISCOM matrix or ISCOM complex particle is Fraction C (a “FractionC Matrix”). Thus, preferred compositions comprise, as an adjuvant, aFraction A Matrix adjuvant and a Fraction C Matrix adjuvant. The amountsof each Matrix in the composition may vary. For example, the amount ofFraction A Matrix may be about 80% (w/w), about 85% (w/w), about 90%(w/w), about 92% (w/w), or about 95%0/(w/w) with the remainder FractionC Matrix. A suitable example of a suitable 85:15 Fraction A Matrix andFraction C Matrix combination is Matrix-M™ (Novavax AB, Uppsala,Sweden), a mixture of Fraction A Matrix and Fraction C Matrix at a ratioof about 85 to about 15.

Other saponin fractions, such as QS-7 and QS-21 fractions, theirproduction and their use is described in U.S. Pat. Nos. 5,057,540;6,231,859; 6,352,697; 6,524,584; 6,846,489; 7,776,343, and 8,173,141.These fractions may be used in the methods and compositions disclosedherein.

Immune Stimulators

Compositions of the disclosure may also be formulated with “immunestimulators.” These are the body's own chemical messengers (cytokines)to increase the immune system's response. Immune stimulators include,but are not limited to, various cytokines, lymphokines and chemokineswith immunostimulatory, immunopotentiating, and pro-inflammatoryactivities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12,IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colonystimulating factor (CM); and other immunostimulatory molecules, such asmacrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc. Theimmunostimulatory molecules may be administered in the same formulationas the compositions of the disclosure, or may be administeredseparately. Either the protein or an expression vector encoding theprotein may be administered to produce an immunostimulatory effect.Thus, in one embodiment, the disclosure comprises antigenic and vaccineformulations comprising an adjuvant and/or an immune stimulator.

Methods of Inducing Immune Responses

Also provided in the present disclosure are methods of eliciting animmune response against pathogens. The method involves administering animmunologically effective amount of a composition comprising a multimerprotein to a subject. Administration of an immunologically effectiveamount of the composition of the disclosure elicits an immune responsespecific for epitopes present on the fusion protein. Such an immuneresponse can include B cell responses and/or T cell responses. Whenadministered to a subject, the multimer proteins preferably induceneutralizing antibodies. Preferably, the immune response includeselements that are specific for at least one conformational epitopepresent each protein contained in the multimer protein.

Administration

Administration may be by any suitable route. Suitable routes includeparenteral administration (e.g., intradermal, intramuscular, intravenousand subcutaneous), epidural, and mucosal (e.g., intranasal and oral orpulmonary routes or by suppositories), transdermally or intradermally.Administration may be by infusion or bolus injection, by absorptionthrough epithelial or mucocutaneous linings (e.g., oral mucous, colon,conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladderand intestinal mucosa, etc.) and may be administered together with otherbiologically active agents. In some aspects, intranasal or other mucosalroutes of administration may result in an antibody or other immuneresponse that is substantially higher than other routes ofadministration. Administration can be systemic or local.

In some aspects, administration may be by injection using a needle andsyringe, by a needle-less injection device. In other aspects,administration is by drops, large particle aerosol (greater than about10 microns), or by spray into the upper respiratory tract.

In some aspects, a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the components of the formulationsis provided. In a particular aspect, the kit may include two containers,a first container containing a multimer protein, and a second containercontaining an adjuvant. Associated with such container(s) may be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. Formulations may also be packaged in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of composition.

In some aspects, administration may be targeted. For example, thecompositions may be administered in such a manner as to target mucosaltissues in order to elicit an immune response at the site ofimmunization. Mucosal tissues such as gut associated lymphoid tissue(GALT) can be targeted for immunization by using oral administration ofcompositions which contain adjuvants with particular mucosal targetingproperties. Additional mucosal tissues can also be targeted, such asnasopharyngeal lymphoid tissue (NALT) and bronchial-associated lymphoidtissue (BALT).

In some aspects, multiple compositions may be administered each havingdifferent collections of antigens. Where more than one multimer proteinis administered, the proteins may be co-administered simultaneously tothe same position of the subject; for example, by injection of materialfrom one or more containers containing multimer proteins. In otheraspects, they may be co-administered sequentially at different siteswithin a short space of time; for example, one administration may be inthe thigh, and a second administration may be in the arm, with bothadministrations occurring within a short period (e.g. up to 30 minutes).

Human clinical studies can be performed to determine the preferredeffective dose for humans by a skilled artisan. Such clinical studiesare routine and well known in the art. The precise dose to be employedwill also depend on the route of administration. Effective doses may beextrapolated from dose-response curves derived from in vitro or in vivotest systems. Dose may be adjusted based on, e.g., age, physicalcondition, body weight, sex, diet, time of administration, and otherclinical factors.

While stimulation of immunity with a single dose is possible, additionaldosages may be administered, by the same or different route, to achievethe desired effect. In neonates and infants, for example, multipleadministrations may be required to elicit sufficient levels of immunity.Administration can continue at intervals throughout childhood, asnecessary to maintain sufficient levels of protection againstinfections. Similarly, adults who are particularly susceptible torepeated or serious infections, such as, for example, health careworkers, day care workers, family members of young children, theelderly, and individuals with compromised cardiopulmonary function mayrequire multiple immunizations to establish and/or maintain protectiveimmune responses. Levels of induced immunity can be monitored, forexample, by measuring amounts of neutralizing secretory and serumantibodies, and dosages adjusted or vaccinations repeated as necessaryto elicit and maintain desired levels of protection.

The vaccine compositions may also be used for preparing antibodiesagainst the toxins useful for passive administration therapies. SeeCasadevall. “Passive Antibody Administration (Immediate Immunity) as aSpecific Defense Against Biological Weapons,” Emerging InfectiousDiseases. 2002; 8(8):833-841.

EXAMPLES Example 1

C. difficile Triple Toxin Vaccine Constructs

Two triple toxin vaccines were constructed. A diagram of the proteinstructures is shown in FIG. 1. Triple toxin 1420 (also referred to asBV1420) contains, from N-terminus to C-terminus, an Activation domainpeptide, a mature CDTb peptide, a TcdB RBD peptide, and a TcdA RBDpeptide containing 19 repeats (RI9). A furin cleavage site (RARRRKKR;SEQ ID NO:27) was located between the activation domain and mature CDTbpeptides. FIGS. 2 and 3 show the protein and genetic sequence of BV1470,respectively. Linker sites at either end of the TcdB peptide.

Example 2 Expression of Triple Toxin Vaccine

Sf9 cells were transformed with a baculovirus vector expressing thetriple vaccine as a single transcript. Expression data from the Sf9cells is shown in FIG. 2. FIG. 2 shows expression of each proteinsharvested at 48 hours and at 72 hours. Remarkably, even though eachprotein is over 200 kDa, high level production is achieved. FIG. 7 showsa time course of expression from 48 hours to 96 hours. The data showsthat, for both proteins, the protein is highly soluble.

Example 3 Purification of Triple Toxin Vaccine

To solubilize and purify the particles, Tergitol (NP9) was directlyadded to cell culture to final concentration of 0.2% NP9/25 mM Tris/50mM NaCl/pH8.0. Incubate at room temperature for 1 hour then centrifugethe lysate at 9000 g for 30 min twice. Collected the supernatantcontaining the nanoparticles. The supernatant is then added to in BufferA and eluted in Buffer B (Buffer A: 25 mM Tris pH 8.0/50 mM NaCl BufferB: 25 mM Tris pH 8.0/1M NaCl). The eluate is appled to Phenyl HP columns(Buffer A: 350 mM Na-Citrate/25 mM Tris pH7.5 and Buffer B: 5 mM TrispH8.0) and then to a Source 30Q column (Buffer A: 25 mM Tris pH8.0/250mM NaCl Buffer B: 25 mM Tris pH8.0/1M NaCl). The pooled fractionscontaining the product are passed through a 2 micron filter. See FIGS.4-6. Purification of 1470 from Sf9 yielded 269 mg/liter of protein.Purification of 1420 from Sf9 cells yielded 166 mg/liter.

Example 4 Analysis of Triple Toxin Vaccine Particles

Particle size distribution by volume graph for triple toxin BV1420 wasanalyzed by dynamic light scattering using a Zeta Sizer Nano. Graph ofsize distribution by volume is shown in FIG. 7. The average diameter was˜30 nm. FIG. 8 shows particle size distribution by intensity graph fortriple toxin BV1470. The average diameter was ˜18 nm.

FIG. 9 shows various electronmicrographs of negative stained tripletoxin BV1420. Electron-micrograph of purified triple toxin BV1420 wasdiluted to approximately 10 ug/ml and negatively stained with uranylacetate.

Example 5

C. difficile Triple Toxin Vaccine: Lethal Toxin Challenge and AnimalSurvival

FIG. 10 provides the results of a mouse trial of the Triple ToxinVaccine against Toxin A and Binary Toxin. Groups 1-6 were administeredBV1420 antigen (30 gig) or PBS as shown. Groups 1 and 4 contain 50 μgAlum OH; Groups 2 and 5 contained 50 μg Alum OH and 50 μg ISCOM Matrix Madjuvant. Mice were immunized at Day 0 and Day 14, with bleeds at Day 0,14, and 32. Mice were challenged with Toxin A or Binary Toxin at Day 35.

FIG. 11 shows serum IgG responses. PBS did not induce antibodies, asexpected. The Triple Toxin Vaccines, either with Alum OH or with bothAlum OJ and Matrix M induced Titers ranging from about 10⁴ to about 10⁶against Toxin A, Toxin B, and CDTb. FIG. 12 establishes that theantibodies neutralized both Toxin A and CTDb. FIG. 13 shows animalsurvival for the 6 groups. Groups 1, 2, 4, and 5 showed 100% survival.Except for two mice in the binary toxin challenge, all the animals inthe control PBS groups died. These data establish that the triple toxinvaccine protects against the effect of the toxins.

In a second challenge study, for Toxin B, several constructs wereproduced and tested alone or in combination. Group 1 mice wereadministered BV1420 (30 μg) with Alum OH. Group 2 mice were administeredBV1470 (30 μg) with Alum OH, Group 3 was administered a tandem proteincontaining rotavirus VP6 and the TcdB RBD (10 μg) with Alum OH. Group 4mice were administered BV1470 and VP6/TcdB RBD. Group 5 was administeredToxoid B (10 μg). Group 6 was the control and was administered PBS.Anti-IgG response is shown in FIG. 15. High titers antibodies wereobtained in each case. Each of the groups containing the Toxin A peptideinduced high titer anti-Toxin A responses ranging between 10⁴ and about10⁵. All groups were administered the Toxin B peptide and eachdemonstrated high titer ranging between 10⁴ and about 10⁶. Each of thegroups containing the Binary Toxin peptide induced high titer responsesranging between 10⁵ and about 10⁶. FIG. 16 establishes that theantibodies were produced that neutralized both Toxin B, with the ToxoidB showing higher levels.

Survival of the Groups 1-6 mice is shown in FIG. 17. All mice in the PBScontrol Group died by Day 3, with 5 of 6 dead within one day. Toxin Bsurvival was 100%. For groups 1 to 4, survival rates ranged from 67% to83%.

Example 6 Additional Triple Toxin Vaccines

Additional vaccines can be produced while obtaining high expressionlevels. FIGS. 18 and 19 shows additional trivalent vaccine proteins withthe TcdB gene translocations gene. BV1512 is shown in the bottomdiagram. FIG. 18 shows additional vaccines structures: Multimer ProteinSequence: Sequence of BV1512 multimer vaccine protein showing CDTbprotein separated from the Translocation Domain (TD) by an A-S linkerand the TD separated from the TcdAR19 portion by an S-R linker. FIG. 19shows expression of the multimer protein BV1512 from Sf9 cells.

Example 7 Quadrivalent Vaccines

Multimer proteins containing four peptides were produced. FIG. 20. Inthis example, a peptide from a second TcdB strain was introduced tobroaden immunity against an additional C. difficile strain. The firstquadrivalent multimer protein (CBAB, or pCDTb/TcdB₆₃₀/TcdAR19/TcdB₀₂₇)included a TcdB peptide from Strain 027 added at the C-terminus (SeeFIG. 20, upper diagram). In a second quadrivalent multimer pepride, aTcdB peptide from Strain 027 peptide was introduced between the TcdBprotein and the TcdA(R19) protein from the first strain, strain 630 (SeeFIG. 20, lower diagram). FIG. 21 shows expression of the CBBAquadrivalent multimer from Sf9 cells as described above. The data showsthat the yield obtained was 42 mg/L. A second protein (CBBA, orpCDTb/TcdB₆₃₀/TcdAR19/TcdB₀₂₇ as shown in FIG. 26) was also produced inthe Sf9 system and achieved 40 mg/L yield. See FIG. 22.

Example 8 Design, Expression, and Purification of T-toxin and Q-toxinFusion Proteins

Chimeric fusion proteins were constructed to encode RBD of C. difficileTcdA, TcdB₍₀₀₃₎, TcdB₍₀₂₇₎, and CDTb. The RBD amino acid sequence forTcdA was derived from C. difficile strain VPI 10463 (ATCC 43255), NCBIP16154 (toxinotype 0, ribotype 003); TcdB₍₀₀₃₎ from strain VPI 10463(ATCC 43255), NCBI P18177 (toxinotype 0, ribotype 003): TcdB₍₀₂₇₎ fromstrain CDI96, NCBI WP_009888442.1 (toxinotype III, ribotype 027); andCDTb from strain CD196, GenBank ABS57477.1 (toxinotype III, ribotype027).

The coding sequences for TcdA RBD (truncated with 19 of 38 repeats),TcdB₍₀₀₃₎ and TcdB₍₀₂₇₎ RBDs (24 repeats each), and CDTb were codonoptimized for expression in insect cells (GenScript).

The nucleotide sequences encoding the CDTb gene fragment (amino acids1-835), TcdA RBD (1314 base pairs [bp], 6816-8130 bp), and TcdB₍₀₀₃₎ RBD(1608 bp, 5493-7098 bp) were obtained by PCR amplification from thesynthesized gene. PCR-amplified gene fragments were digested withrestriction enzyme: CDTb with BamHI/NheI; TcdB₍₀₀₃₎ RBD with NheI/XbaI;and TcdA RBD with XbaI/HindIII. After digestion, the three genes wereligated into the BamH1 and HindIII sites of pFastBac1 (Invitrogen). Theplasmid encoding the three RBDs was used to construct a recombinantAutographa californica Multiple Nuclear Polyhedrosis Virus (AcMNPV)baculovirus using the Bac-to-Bac baculovirus expression system(Invitrogen) in Spodoptera frugiperda (Sf9) insect cells to express thetrivalent fusion protein, hereafter referred to as T-toxin (FIG. 23B).

TcdB₍₀₂₇₎ RBD (1608 bp, 5493-7098) digested with Spel/HinIII was fusedto the C-terminus of the trivalent fusion gene to form the plasmid andbaculovirus construct encoding the RBD of all four toxins, which wassimilarly expressed in Sf9 cells to produce the quadravalent fusionprotein, hereafter referred to as Q-toxin (FIG. 23B; SEQ ID NO: 21).

The construct thus contains pCDTb: 835 amino acid from 1-835; Strain:CD196; toxinotype: III, ribotype: 027; GenBank: ABS57477.1;TcdBoo₃: 536amino acid from 838-1373; NCBI: P18177, STRAIN=ATCC 4325/VPI 10463,Toxinotype 0, Ribotype: 087; TcdA: 438 amino acid from 1376-1813; NCBI:P16154, STRAIN=ATCC 4325/VPI 10463, Toxinotype 0; Ribotype: 087, andTcdB₀₂₇: 536 amino acid from 1815-2351; NCBI: 013315, strain CD196;toxinotype: HI, ibotype: 027. Each of the portion is separated by a twoamino acid linker: AS between the pCDTb portion and the TcdB003 portion,SR between the TcdB003 portion and the TcdA portion, TS between the TcdAportion and the TcdB027 portion.

Fusion proteins were extracted by detergent lysis in a buffer comprising0.2% Tergitol NP-9 in 25 mM Tris buffer (pH 8.0), 250 mM NaCl and 2μg/mL leupeptin. Lysates were purified by centrifugation, and the fusionproteins were purified with Fractogel EMD TMAE, phenyl HP and 30Q columnchromatography. Purified T-toxin and Q-toxin were formulated in 25 mMTris and 250 mM NaCl (pH 8.0) at approximately 4.0 mg/mL and stored at<−60° C. Recovery of purified T-toxin and Q-toxin was 267 and 154 mg/L,respectively. T-toxin and Q-toxin migrate in SDS-PAGE gels withmolecular weights of 205 kDa and 268 kDa, respectively, and purityof >90% (FIG. 23A). Western blot analysis with toxin-specific antibodiesconfirmed expression of CDTb, TcdB, and TcdA in each fusion protein(FIG. 23B-D).

Example 9 Immunogenicity of T-toxin and Q-toxin Fusion Proteins in Mice

To evaluate immunogenicity of T-toxin and Q-toxin fusion proteins, Mousestudies were conducted in accordance with Noble Life Sciences'Institutional Animal Care and Use Committee (IACUC) approved protocols.Female C57BL/6 mice (6-8 weeks old) were immunized IM on Days 0 and 14with T-toxin (30 or 100 pig) or Q-toxin (100 pig) formulated with 50 μgaluminum hydroxide (alum), or PBS (control). Serum was collected 18 daysafter the second dose. Mice were challenged intraperitoneally (IP) 3weeks after the second immunization with a 100% minimal lethal dose(MLD_(100%)) of TcdA, TcdB₍₀₀₃₎, or CDTa and CDTb.

Mouse sera was evaluated for antibodies to the toxins by ELISA. A96-well MaxiSorp microtiter plates (Thermo Scientific) were coated witheach toxin (2 μg/mL) overnight at 2-8° C. Five-fold serial dilutions ofsera were added to plates in duplicate. Bound antibodies were detectedwith horseradish peroxidase-conjugated goat anti-mouse IgG (SouthernBiotech). 3,3′,5, 5′-tetramethylbenzidine (TMB) substrate (Sigma) wasadded and the reaction stopped with TMB Stop Buffer (ScytekLaboratories). Plates were read at 450 nm with a SpectraMax Plus platereader (Molecular Devices); results were analyzed using SoftMax Prosoftware. Titers were reported as the reciprocal dilution that resultedin a reading of 50% the maximum OD_(450 nm). Titer values recorded asbelow the lower limit of detection (LLOD) were assigned a titer 50 forcalculating GMT. Mouse serum IgG titers following immunization were highfor TcdA, TcdB, and CDT and comparable between T-toxin and Q-toxin (FIG.25A).

Vero cells (CCL-81, ATCC) were maintained in DMEM supplemented with 20%heat-inactivated fetal bovine serum (FBS) and antibiotics (Gibco).Two-fold serial dilutions of mouse sera were prepared in 96-well,flat-bottom tissue culture plates (Thermo Scientific). An equal volume(50 μL) of assay medium (1×DMEM with 5% heat-inactivated FBS, 1×NEAA,0.3% dextrose, 1× penicillinistreptomycin/glutamine, 0.006% Phenol Red)containing 2× minimum cytotoxic dose of TcdA, TcdB, or CDT was added todiluted serum and incubated for 1 hour at 37° C. Vero cells (7.5×10⁴cells/mL) suspended in 50 μL medium and 150 μL sterile mineral oil(Sigma) were added and plates were incubated for 6-7 days at 37° C.After incubation, plates were observed for well color. Media andtoxin-treated control wells were red/reddish-pink; cell control wellswere yellow/yellow-orange. For each sample dilution, the last well thatwas yellow/yellow-orange was recorded as the endpointneutralizing-antibody titer. Titer values recorded as <LLOD wereassigned a value of 5 for calculating GMT. Toxin-neutralizing antibody(TNA) titers to each of the three toxins were comparable between theT-toxin and Q-toxin fusion proteins (FIG. 25B).

Three weeks after the second immunization, mice were challengedTcdB₍₀₀₃₎. The group vaccinated with Q-toxin had 80% survival(p=0.0043), while 65% (p=0.018) of the T-toxin group survived challenge.In contrast, only 20%0 survived toxin challenge in the control group(FIG. 25C).

Example 10 Immunogenicity of T-toxin and Q-toxin Fusion Proteins inHamsters

Golden Syrian hamsters (HsdHan:Aura; Harlan Laboratories), males aged5-7 weeks and 70 to 100 grams, received 3 immunizations at 3-weekintervals with 30 μg Q-toxin and 120 μg alum, or PBS (control),administered IM in alternating thighs. Two weeks after the thirdimmunization serum was collected and animals were treated with 10 mg/kgclindamycin IP. One day later, animals were challenged by gavage withstrain 630 or NAP1 and were observed for 8 days.

Hamster sera was evaluated for antibodies to the toxins by ELISA. A96-well MaxiSorp microtiter plates (Thermo Scientific) were coated witheach toxin (2 μg/mL) overnight at 2-8° C. Five-fold serial dilutions ofsera were added to plates in duplicate. Bound antibodies were detectedwith horseradish peroxidase-conjugated rabbit anti-hamster IgG (SouthernBiotech). 3,3′,5, 5′-tetramethylbenzidine (TMB) substrate (Sigma) wasadded and the reaction stopped with TMB Stop Buffer (ScytekLaboratories). Plates were read at 450 nm with a SpectraMax Plus platereader (Molecular Devices); results were analyzed using SoftMax Prosoftware. Titers were reported as the reciprocal dilution that resultedin a reading of 50% the maximum OD_(450 nm). Titer values recorded asbelow the lower limit of detection (LLOD) were assigned a titer 50 forcalculating GMT. Hamsters immunized thrice at 3-week intervals withQ-toxin produced high IgG titers to the TcdA, TcdB, and CDTb toxins(FIG. 26A).

Vero cells (CCL-81, ATCC) were maintained in DMEM supplemented with 20%heat-inactivated fetal bovine serum (FBS) and antibiotics (Gibco).Two-fold serial dilutions of hamster sera were prepared in 96-well,flat-bottom tissue culture plates (Thermo Scientific). An equal volume(50 μL) of assay medium (1×DMEM with 5% heat-inactivated FBS, 1×NEAA,0.3% dextrose, 1× penicillin/streptomycin/glutamine, 0.006% Phenol Red)containing 2× minimum cytotoxic dose of TcdA, TcdB, or CDT was added todiluted serum and incubated for 1 hour at 37° C. Vero cells (7.5×10⁴cells/mL) suspended in 50 μL medium and 150 μL sterile mineral oil(Sigma) were added and plates were incubated for 6-7 days at 37° C.After incubation, plates were observed for well color. Media andtoxin-treated control wells were red/reddish-pink; cell control wellswere yellow/yellow-orange. For each sample dilution, the last well thatwas yellow/yellow-orange was recorded as the endpointneutralizing-antibody titer. Titer values recorded as <LLOD wereassigned a value of 5 for calculating GMT. TNA titers to each of thethree toxins were comparable between the T-toxin and Q-toxin fusionproteins (FIG. 31B).

After clindamycin treatment, animals infected with C. difficile strain630 had 90% survival (FIG. 26C), while animals infected with NAP1 had75% survival (FIG. 31D). All animals in the placebo group died within48-72 hours following infection with either strain.

INCORPORATION BY REFERENCE

Each of the patents and published applications identified herein areincorporated herein for all purposes.

1. A multivalent immunogenic polypeptide comprising portions of at leastfour C. difficile toxin proteins.
 2. The multivalent immunogenicpolypeptide of claim 1, wherein the portions are selected from the groupof toxins consisting of Binary toxin (CDT), Toxin A (TcdA) protein andToxin B (TcdB).
 3. The multivalent immunogenic polypeptide of claim 2,wherein the portions are from a CDT protein, a Toxin A protein and twoToxin B proteins, wherein the Toxin B proteins are from distinct C.difficile strains.
 4. The multivalent immunogenic polypeptide of claim3, wherein at least one Toxin B portion is from an epidemic strain. 5.The multivalent immunogenic polypeptide from claim 4, wherein theepidemic strain is the NAP1 strain (TcdB₍₀₂₇₎).
 6. The multivalentimmunogenic polypeptide of claim 3, wherein one of the Toxin B portionis from 630 strain (TcdB₍₀₀₃₎).
 7. The multivalent immunogenicpolypeptide of claim 3, wherein the portions are oriented with the ToxinA portion between the two Toxin B portions.
 8. The multivalentimmunogenic polypeptide of claim 3, wherein the CDT portion isN-terminal to one or both of the Toxin B portions.
 9. The multivalentimmunogenic polypeptide of claim 3, wherein the CDT portion has an aminoacid sequence that comprises or consists of SEQ ID NO:22 or an aminoacid sequence having at least 90% homology to the sequence.
 10. Themultivalent immunogenic polypeptide of claim 3, wherein one of the ToxinB portions has an amino acid sequence that comprises or consists of SEQID NO-23 or an amino acid sequence having at least 90% homology to thesequence.
 11. The multivalent immunogenic polypeptide of claim 3,wherein the second Toxin B portion has an amino acid sequence thatcomprises or consists of SEQ ID NO:25 or an amino acid sequence havingat least 90% homology to the sequence.
 12. The multivalent immunogenicpolypeptide of claim 3, wherein the Toxin A portion has an amino acidsequence that comprises or consists of SEQ ID NO:24 or an amino acidsequence having at least 90% homology to the sequence.
 13. Themultivalent immunogenic peptide of claim 1 wherein the portions areseparated by a two amino acid linker, a three amino acid linker, or afour amino acid linker.
 14. The multivalent immunogenic peptide of claim13 wherein the portions are separated by a two amino acid linker and thelinker is selected from the group consisting of Alanine-Serine (AS),Leucine-Glutamic acid (LE), and Serine-Arginine (SR).
 15. Themultivalent immunogenic polypeptide of claim 1 wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:21.
 16. A nucleic acidmolecule comprising a polynucleotide encoding the polypeptide of any ofclaims 1 to
 15. 17. A method of preparing the polypeptide of claim 1comprising (a) expressing the polypeptide in an insect host cell, (b)purifying the polypeptide in the presence of a non-ionic detergent inthe form of a nanoparticle, and (c) suspending the nanoparticle in apharmaceutically acceptable carrier, excipient, or diluent.
 18. Themethod of claim 17, wherein the an insect host is an Sf9 cell.
 19. Themethod of claim 17 or claim 18, wherein the insect host cell istransfected with a recombinant baculovirus construct under suitableconditions for expression of the polypeptide.
 20. A vaccine compositioncomprising the immunogenic polypeptide of any of claims 1 to 15, and apharmaceutically acceptable carrier, excipient, or diluent.
 21. Thevaccine composition of claim 20 wherein the composition comprises anadjuvant.
 22. The vaccine composition of claim 20 or 21 wherein theadjuvant is a saponin-based adjuvant.
 23. The vaccine composition of anyof claims 20 to 22 wherein the saponin-based adjuvant contains FractionA Matrix and Fraction C Matrix.
 24. The vaccine composition of any ofclaims 20 to 23 wherein the amount of Fraction A Matrix is about 85% toabout 92% and the remainder is Fraction C Matrix.
 25. Use of apolypeptide according to any of claims 1 to 15 or the nucleic acidaccording to claim 16, for the manufacture of a medicament for theprevention or treatment of Clostridium Difficile infection.
 26. Ananoparticle comprising the multivalent immunogenic polypeptide of anyof claims 1 to 15 and a non-ionic detergent.